The structure of chromatin is tightly regulated by the well choreographed actions of multiple elements. Disruption of the normal pattern of chromatin often leads to mis-regulation of gene expression, a common feature of cancer and other human diseases. Therefore, it is important to understand the detailed mechanism regulating chromatin dynamics. The long-term objective of this proposal is to understand how chromatin- related complexes help the transcription machinery overcome the nucleosomal barriers while still maintaining genome integrity. Recently, we and others discovered a novel signaling pathway through which RNA polymerase II (Pol II) maintains chromatin integrity while transcribing through nucleosomal templates. A histone methyltransferase Set2 binds the phosphorylated CTD of elongating Pol II and co-transcriptionally methylates histone H3K36, which is then recognized by a histone deacetylase complex, Rpd3S. Once targeted, Rpd3S deacetylates transcribed regions to preserve the accuracy of transcription initiation, thus restricting transcription to bonafide promoters but not cryptic transcription start sites. This proposal is intended to dissect the detailed mechanisms driving this crucial pathway via three specific aims. (1) Analysis of Rpd3S binding to K36 methylated nucleosomes and its implication in transcription elongation. We will test how multiple domains within Rpd3S coordinate to achieve synergistic binding. (2) Dissecting the molecular mechanism by which Pol II exploits K36 methylation as a marker for short term transcription memory. We will examine if elongating Pol II can control the directionality of relevant histone modifications around the transcription fork. (3) Identification of the temporal control mechanism of K36 methylation during transcription elongation. We will explore the roles of histone demethylases and the signals for removal of this reversible histone modification. Importantly, human Set2 has been shown to interact with Huntingtin, the Huntington disease protein; and the treatment of histone deacetylase inhibitors can arrest neurodegeneration in a model system. Therefore, our understanding on the Set2- Rpd3S pathway may also help design potential therapeutic agents to treat neurodegenerative diseases.